Abstract:

A method for promoting differentiation of clonogenic neural stem cells
(NSCs), comprising administering to a patient in the need of such
promoting a coumarin compound represented by formula I or by formula II.
The representative coumarin compounds include 7-hydroxycoumarin,
daphnoretin, scopoletin, edgeworin, aesculetin and
esculetin-6-β-D-glucopyranoside. The coumarin compounds showed
significant activity of directing the differentiation of NSCs in
pharmacological test and thereof could be used to prepare drugs to direct
NSCs differentiated to oligodendrocyte progenitor cells (OPCs) for the
treatment of demyelinating diseases or spinal cord injury. The drug could
be a pure coumarin compound or a pharmaceutical composition comprising a
therapeutical dose of a coumarin compound as active ingredients and a
pharmaceutically-acceptable carrier. The content of the active
ingredients in the pharmaceutical composition is between 0.1% and 99.5%
by weight.

Claims:

1. A method for promoting differentiation of clonogenic neural stem cells
(NSCs), comprising administering to a patient in the need of such
promoting a coumarin compound represented by formula I or by formula II,
##STR00023## whereinR1 at each occurrence represents hydrogen,
hydroxyl, phenyl, or a glycosidic moiety comprising 1-5 sugars.

2. A method for promoting differentiation of clonogenic neural stem cells
(NSCs), comprising administering to a patient in the need of such
promoting a pharmaceutical composition comprising an excipient and a
coumarin compound represented by formula I or by formula II, ##STR00024##
whereinR1 at each occurrence represents hydrogen, hydroxyl, phenyl,
or a glycosidic moiety comprising 1-5 sugars.

3. The method of claim 2, wherein said pharmaceutical composition
comprises between 0.1% and 99.5% of said coumarin compound by weight.

6. The method of claim 1, wherein said coumarin compound administered to a
patient has been prepared by extraction from a plant or by chemical
synthesis

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]This application is a continuation of International Patent
Application No. PCT/CN2007/002638 with an international filing date of
Sep. 3, 2007, designating the United States, now pending, and further
claims priority benefits to Chinese Patent Application No.
200610030917.1, filed on Sep. 7, 2007. The contents of these
specifications are incorporated herein by reference.

[0005]Clonogenic neural stem cells (NSCs) are self-renewing cells that
maintain the capacity to differentiate into brain specific cell types,
such as neurons, astrocytes, and oligodendrocytes, and may also replace
or repair diseased brain tissue. The neural stem cells are abounding in
the fetal or adult spinal cord and in the third and fourth ventricles.
These cells persist in the subventricular zone, hippocampus, cortex, and
spinal cord, even in the adult.

[0006]Neural stem cells (NSCs) are immature cells with the ability to
renew themselves and give rise to neurons, astrocytes, and
oligodendrocytes. Isolated NSCs are able to proliferate in response to
basic fibroblast growth factor or epidermal growth factor, and when the
culture conditions are altered, they differentiate into several
phenotypes of neurons. Furthermore, neurons derived from NSCs form
functional synapses in vitro and in vivo. These results suggest that NSCs
have the potential to differentiate into appropriate neurons to form a
functional neuronal circuitry.

[0009]However, spontaneous differentiation of neural stem cells (NSCs) is
generally inefficient and mainly leads to large amount of astrocytes,
which are not useful for cell-based therapy (Cao et al. 2001).

[0010]Demyelination is a prominent feature of many CNS disorders,
including multiple sclerosis (MS), spinal cord injury (SCI) and stroke
(Mason et al. 2004, Tanaka et al. 2003, Keirstead et al. 2005). The
currently available treatments aim at preventing future demyelination
based on immunomodulation or immunosuppression (Bernd et al. 2005), and
have so far been only partly successful by reducing disease progression
without stopping further demyelination (Chari and Blakemore 2002). Since
the primary defect appears to be demyelination, the most straightforward
approach is to provide new myelinating cells for compensation within the
lesions (Groves et al. 1993). Given the greater mitotic and migratory
potential of oligodendrocyte progenitor cells (OPCs), OPCs are the
preferred cell for myelin repair (Chari and Blakemore 2002).

[0011]Although human OPCs have been isolated from both abortion-derived
and adult human CNS (Aloisi, et al. 1992, Armstrong, et al. 1992), it is
obviously difficult to acquire sufficient donor cells and ethically
unlikely ever to be acceptable. OPC cell lines as an unlimited cell
source have been generated and shown to remyelinate experimental lesions
(Barnett, et al. 1993), but uncontrolled cell proliferation of a cell
line could be a limiting factor for a therapeutic strategy based on cell
transplantation.

[0012]Clearly, more efficient and selective approaches are needed to
direct the differentiation of NSCs, to produce homogenous populations of
OPCs.

SUMMARY OF THE INVENTION

[0013]In view of the above-described problems, it is one objective of the
invention to provide methods for directing the differentiation of
clonogenic neural stem cells (NSCs).

[0014]It is another objective of the invention to provide a method for
treating demyelinating diseases or spinal cord injury.

[0015]It is another objective of the invention to provide a pharmaceutical
composition comprising a carrier and a coumarin compound of the formula I
or II as active ingredients.

[0018]The coumarins disclosed in the invention are distributed in many
plant species. These coumarins could be obtained by extraction from
plants or by chemical synthesis.

[0019]The representative compounds of the coumarins disclosed in this
invention include, without limitation, 7-hydroxycoumarin, daphnoretin,
scopoletin, edgeworin, aesculetin, and
esculetin-6-β-D-glucopyranoside.

[0020]Previously, we purified and identified more than 2,000 natural
compounds from Traditional Chinese Medicine (TCM) and built up a natural
products library. The high-throughput screening of the library lead to
the discovery of the coumarins disclosed in the invention which exhibit
the activity of directing differentiation of clonogenic neural stem cells
(NSCs). Further studies demonstrated that these coumarin compounds could
promote NSCs differentiation to oligodendrocyte precursor cells (OPCs).

[0021]In certain embodiments of the present invention, the pharmaceutical
composition disclosed herein comprises a therapeutical dose of a coumarin
compound as active ingredient and a pharmaceutically acceptable carrier.
The content of the active ingredient is between 0.1% and 99.5% by weight.

[0022]In certain embodiments of the present invention, the compounds and
the pharmaceutical compositions disclosed herein are administered to a
patient to direct NSCs differentiation to OPCs whereby treating a
demyelinating disease or spinal cord injury.

[0024]In certain embodiments of the present invention, the compounds and
pharmaceutical compositions disclosed herein are administered to a
patient in need of such treatment orally, intranasally, rectally, or
parenterally. When given orally, the compounds or pharmaceutical
compositions can be prepared in tablets, powder, granule, gelatin
capsule, or liquid preparations (water suspension, oil suspension, syrup,
elixir, etc.). When given parenterally, the compounds or pharmaceutical
compositions can be prepared in solution, water suspension, or oil
suspension for injection. The preferred form is tablet, coated tablet,
gelatin capsule, suppository, nasal spray, or injection.

[0025]In certain embodiments of the invention, the dosage forms of the
compounds and pharmaceutical compositions disclosed herein can be
prepared by conventional methods used in the pharmaceutical industry.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026]Detailed description will be given below with reference to
accompanying drawings, in which:

[0027]FIG. 1 is a cell morphogram of NSCs treated with 7-hydroxycoumarin;

[0028]FIG. 2 is a cell morphogram of NSCs treated with daphnoretin; and

[0029]FIG. 3 is a cell morphogram of NSCs treated with DMSO.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0030]Examples of preparations and results of pharmacological test
according to the present invention are shown below. The Examples do not
limit the scope of the invention, but are intended to make the invention
more clearly understandable.

[0031]Preparation of Compounds:

Example 1

7-hydroxycoumarin

##STR00003##

[0033]Roots of Daphne giraldii Nitsche (11 kg) were air-dried and chopped
into small pieces. Ethanol extract of the roots (95% v/v) was prepared
and evaporated in vacuo. The residue was suspended in water, and then
partitioned with petroleum ether, chloroform (CHCl3), ethyl-acetate
(EtOAc) and n-butyl alcohol (n-BuOH). The four fractions were
concentrated in vacuo and stored at -20° C. prior to further
purification. The EtOAc fraction was subjected to silica gel
chromatography. The eluents for this fraction on column chromatography on
silica gel were different ratios of CHCl3 and MeOH, as mobile phase
to afford nine sub-fractions. Sub-fraction 2 was further purified by
silica gel chromatography to afford a single compound (white powder). The
compound was identified as 7-hydroxycoumarin using UV, IR, mass, and NMR
spectra.

Example 2

Daphnoretin

##STR00004##

[0035]Roots of Daphne giraldii Nitsche (11 kg) were air-dried and chopped
into small pieces. The ethanol extract of the roots (95%, v/v) was
prepared and evaporated in vacuo. The residue was suspended in water, and
then partitioned with petroleum ether, chloroform (CHCl3),
ethyl-acetate (EtOAc) and n-butyl alcohol (n-BuOH). The four fractions
were concentrated in vacuo and stored at -20° C. prior to further
purification. The EtOAc fraction was subjected to silica gel
chromatography. The eluants for this fraction on column chromatography
over silica gel were different ratios of CHCl3 and MeOH, as mobile
phase to afford nine sub-fractions. Sub-fraction 5 was further purified
on silica gel chromatography to afford a single compound (yellow powder).
The compound was identified as daphnoretin using UV, IR, mass, and NMR
spectra.

Example 3

Scopoletin

##STR00005##

[0037]Roots of Daphne odora Thunb. var. atrocaulis Rehd. (5 kg) were
air-dried and chopped into small pieces. The roots were then percolated
with ethanol (75%, v/v). The ethanol extract was evaporated in vacuo. The
residue was suspended in water, and then partitioned with petroleum
ether, chloroform (CHCl3), ethyl-acetate (EtOAc) and n-butyl alcohol
(n-BuOH). The four fractions were concentrated in vacuo and stored at
-20° C. prior to further purification. The CHCl3 fraction was
subjected to column chromatography on silica gel, Sephadex LH-20 and HPLC
to afford 12 compounds. One of these compounds (yellow powder) was
identified as scopoletin using UV, IR, mass, and NMR spectra.

Example 4

Edgeworin

##STR00006##

[0039]Roots of Edgeworthia chrysantha (5 kg) were air-dried and chopped
into small pieces. Ethanol extract (80% v/v) was prepared and evaporated
in vacuo. The residue was suspended in water, and then partitioned with
petroleum ether, chloroform (CHCl3), ethyl-acetate (EtOAc) and
n-butyl alcohol (n-BuOH). The four fractions were concentrated in vacuo
and stored at -20° C. prior to further purification. The
CHCl3 fraction was subjected to column chromatography on silica gel,
Sephadex LH-20 and HPLC to afford 12 compounds. One of the compounds
(yellow powder) was identified as edgeworin using UV, IR, mass, and NMR
spectra.

Example 5

Aesculetin

##STR00007##

[0041]The air-dried and powdered stem bark of Fraxinus rhynchophylla
Hance. (15 kg) was extracted 3 times with 40 L ethanol each time for 2 h.
The solvent was evaporated in vacuo. The residue was suspended in water,
and then partitioned with petroleum ether, chloroform (CHCl3),
ethyl-acetate (EtOAc) and n-butyl alcohol (n-BuOH). The four fractions
were concentrated in vacuo and stored at -20° C. The EtOAc extract
was subjected to column chromatography on silica gel (200-300 mesh) and
eluted successively with gradient CHCl3-MeOH mixtures of increasing
polarity to give five compounds. One of the compounds was identified as
aesculetin using UV, IR, mass, and NMR spectra.

Example 6

Esculetin-6-β-D-glucopyranoside

##STR00008##

[0043]The air-dried and powdered stem bark of Fraxinus rhynchophylla
Hance. (15 kg) was extracted three times with 40 L of ethanol, each time
for 2 h. The solvent was evaporated under vacuum. The residue was
suspended in water, and then partitioned with petroleum ether, chloroform
(CHCl3), ethyl-acetate (EtOAc) and n-butyl alcohol (n-BuOH). The
four fractions were concentrated in vacuo and stored at -20° C.
The EtOAc extract was subjected to column chromatography on silica gel
(200-300 mesh) and eluted successively with gradient CHCl3-MeOH
mixtures of increasing polarity to give five compounds. One of the
compounds was identified as esculetin-6-β-D-glucopyranoside using
UV, IR, mass and NMR spectra.

[0050]The active ingredient is mixed with lactose and amylum maydis. The
mixture is wetted by water uniformly. The wetted mixture is filtered,
dried and filtered again. Magnesium stearate is added in the mixture
before tabletting. There is 20 mg active ingredient in each tablet of 250
mg.

[0054]Rat NSCs isolation and culture were performed as described by Weiss
et al (Weiss et al., 1996). Briefly, the cortex of 1- to 2-day-old
newborn SD rats were removed and placed into sterile chilled Hank's
Balanced Salt Solution. After the meninges were carefully removed,
tissues were cut into tiny pieces and gently triturated with a Pasteur
pipette at least 10-15 times. The suspended tissue was centrifuged at 500
g for 5 min and the pellets were re-suspended in a proliferation medium
(DMEM/F12 nutrient (1:1) with additional 2% B27 (Invitrogen Corporation,
Carlsbad, Calif.) supplement and 20 ng/mL bFGF (PeproTech, Rocky Hill,
N.J.)). The cells were plated at a density of 50 cells/mL in non-coated
30 cm flasks. Every 4 days, bFGF was added along with a partial medium
change. After 7 days in vitro, cells formed floating neurospheres. To
subculture NSCs, the neurospheres were centrifuged at 600 g for 5 min
when the diameters of neurospheres reached a size of approximately
100-200 μm; they were then re-suspended in fresh medium and
mechanically dissociated into single cells. Single cells subsequently
were seeded into proliferation medium at a density of 10 cells/mL per
flask. This procedure produced a second generation of neurospheres, and
additional generations of NSCs were produced using this same procedure.

[0055]Compound Screens.

[0056]Neurospheres were plated into 12-well plates on poly-D-lysine coated
35 mm dishes at a density of 10-20 spheres per dish in 1.5 mL medium. 1
hour after plating, compounds were added at a final concentration 100
μM unless otherwise indicated. After 5 days, cells were examined with
Phase contrast image or immunocytochemistry.

[0057]Immunocytochemistry and Cell Count Assessment

[0058]Identification of the different kinds of cells was performed using
indirect immunocytochemistry. Cells on dishes were fixed with 4%
paraformaldehyde at room temperature for 20 min and washed three times in
succession with 0.01 M PBS for 5 min each time. Cells were then treated
with 0.3% Triton 100 containing 10% normal goat serum at room temperature
for 30 min (Xu et al., 2006). Cells were then incubated with primary
antibodies at 4° C. for 12-16 h and washed three times with 0.01 M
phosphate buffered saline (PBS) for 5 min each time. FITC-conjugated
secondary antibodies were added to the cells, and the cells were
incubated at 37° C. for 40 min. After three 5 min PBS washes,
Hochest 33258 nuclear stain was added at room temperature for 10 min,
followed by two more 5 min PBS rinses. The dishes were examined by a
fluorescence microscopy. The primary antibodies included anti-GFAP
(1:200; rabbit; Promega), anti-nestin (1:500; Mouse; Chemicon);
anti-NG2(1:200; Mouse; Chemicon), anti-MBP (1:50; Mouse; Chemicon),
anti-04 (1:100; Mouse; Chemicon), anti-RIP (1:100; Mouse; Chemicon),
anti-olig2 (1:100; rabbit; Abcam). The secondary antibodies were
FITC-labeled antibodies to rabbit IgG (1:200) and TRITIC-labeled
antibodies to mouse IgG (1:200). Immunostained preparations were examined
with an IX70 Olympus microscope equipped for phase contrast and
epi-fluorescence. Light from a 75 W xenon arc lamp (AH2-RX, Olympus)
passed through filter sets (Chroma) to generate excited light for Hochest
33258, FITC and TRITIC stains. All image acquisitions were finished with
computer-controlled CCD (Micromax 5 MHz system, Roper Princeton
Instruments) and MetaMorph Imaging System version 3.6 (Universal Imaging
Corporation). To determine the number of cells expressing a particular
antigen, 100 fields per sample were examined and totaled. Observers
without knowledge of the treatment condition counted the numbers of
positive cells for NG2, compared to total numbers of progeny of NSCs
determined by counting Hochest 33258 staining cell nuclei. Results are
given as mean±S.D. of data for six samples from three independent
experiments. Statistical analyses were carried out using Student's t-test
with SPSS software (Version 10.1).

[0061]NSCs are generally considered as tri-potent, self-renewing
progenitors that can generate neurons, astrocytes and oligodendrocytes,
the three major cell types of the CNS (Weiss et al., 1996). To identify
small molecules that can induce the selective differentiation of NSCs, we
carried out a screen based on a compound library purified from
traditional Chinese herbs. To carry out the primary screen, NSCs were
treated with 100 μM (final concentration) compounds after plating in
neurobasal with 2% B27. After 5 days, cultures were examined under phase
contrast microscope.

[0062]We found that daphnoretin can markedly enhance the generation of
round bipolar or multipolar morphology cells (data not shown), probably
OPCs. To further confirm the effect of daphnoretin, Nestin positive
second-generation neurospheres was plated on cell culture dish and
cultured for 10 days with 100 μM daphnoretin. Cells that migrated out
of the neurosphere and displayed a bipolar or multipolar morphology
characteristic of OPCs, and this characteristic will keep for another 10
days (data not show). We next performed immunostaining for these cells
with NG2 (a specific marker for OPCs) and GFAP (a specific marker for
astrocytes), and the results showed that daphnoretin treatment
dramatically increased the generation of NG2 positive cells. NG2 positive
cells were 21.91% of the total cells treated with DMSO, whereas NG2
positive cells were 65±5.3%, 87±7.5%, 92±8.6%, 95±6.9% when
treated with daphnoretin in 1 μM, 10 μM, 100 μM, and 1 mM,
respectively.

[0063]While particular embodiments of the invention have been shown and
described, it will be obvious to those skilled in the art that changes
and modifications may be made without departing from the invention in its
broader aspects, and therefore, the aim in the appended claims is to
cover all such changes and modifications as fall within the true spirit
and scope of the invention.